1. Field of the Invention
The present invention relates to a process for producing sporangia of microorganisms belonging to Bacillus popilliae containing spores and parasporal bodies useful as a control agent of Scarabaeidae insects (“sporangia containing spores and parasporal bodies” may simply be referred to as “sporangia”) by culturing Bacillus popilliae in liquid medium.
2. Description of the Related Art
The larva of Scarabaeidae insects feed on a wide range of plant roots such as those of grasses, agricultural and horticultural crops and trees, and are known to cause considerable damage. Since these larva live underground, it is difficult obtain control effects by spraying agricultural chemicals from the air, and it is difficult to identify the locations where these larva are present. Therefore, it has been necessary to spray large amounts of agricultural chemicals over a wide range to enable the chemicals to penetrate into the ground, and since there are concerns over detrimental effects on both the natural environment and people, a more effective control method is desired.
Microorganisms belonging to Bacillus popilliae are known to parasitically cause milky disease in the larva of Scarabaeidae insects, and eventually cause their death. Consequently, attempts have long been made to use the sporangia of these microorganisms to control Scarabaeidae insects on which agricultural chemicals have little effect.
For example, an example of a production process is described in Japanese Unexamined Patent Application, First Publication No. 2001-149066 in which a sporangia formation rate (ratio of number of sporangia to number of microbial cells) of 4.8% is obtained by culturing Bacillus popilliae in solid medium containing 0.05–0.5% by weight of activated carbon. However, culturing methods using solid medium have the problem of low productivity.
Various studies have been conducted on culturing methods using liquid medium in order to solve this problem of the aforementioned culturing method using solid medium. For example, Haynes, et al. reported an example of attempting to culture Bacillus popilliae NRRL B-2390S in liquid medium containing 0.5% peptone, 1.5% yeast extract, 0.3% dipotassium hydrogenphosphate, 0.1% glucose and 1% activated carbon (Journal of Invertebrate Pathology, Vol. 22, p. 377–381, 1973). However, only a maximum of 2.06×107 sporangia per 1 ml of liquid culture were obtained, thus resulting in the problem the concentration of sporangia being too low to achieve higher productivity.
In addition, Haynes, et al. also reported that 3.1×107 sporangia per 1 ml of liquid culture were obtained by culturing mature cells of Bacillus popilliae NRRL B-2309S in the late logarithmic increase stage in liquid medium containing 0.5% peptone (tryptone), 1.5% yeast extract, 0.3% dipotassium hydrogenphosphate, 0.1% glucose and 1% activated carbon (Journal of Invertebrate Pathology, Vol. 19, p. 125–130, 1972). However, this culturing method has a long culturing time, taking roughly two weeks.
Moreover, an example of having obtained 1×109 sporangia per 1 ml of liquid culture by culturing in liquid medium containing 1% soluble starch, 0.1% trehalose, 0.5% yeast extract, 0.3% dipotassium hydrogenphosphate and 0.1% calcium carbonate is indicated in U.S. Pat. No. 4,824,671. However, there were no parasporal bodies present in the sporangia, and as a result, the rate of infection with milky disease when sporangia were sprayed at the rate of 2.0×1012 sporangia per 1 kg of soil and allowed to be orally ingested by larva of Scarabaeidae insects was 47.50% after the passage of 7 weeks, indicating weak insecticidal effects on larva of Scarabaeidae insects even when compared with sporangia containing parasporal bodies formed within the bodies of the larva.